A major goal of neuroscience is to understand how cells in the brain communicate with each other. Astrocytes, a type of glial cell, can perceive neuronal activity at synapses and in return release transmitters (gliotransmission) onto neurons to modulate synaptic transmission. An optogenetic approach will be developed that uses light to specifically inactivate the function of a protein required for vesicular release, either VAMP2 or VAMP3. This tool is called STING (Spatio-Temporal INactivation of Gliotransmission) and will be used to determine the role of astrocytic D-serine in modulating NMDAR (N-methyl D-aspartate receptor) function and synaptic plasticity. D-serine, which acts as the endogenous co-agonist of the NMDAR, is critical for inducing long-term potentiation, a form of synaptic strengthening thought to be the cellular basis of learning and memory. We hypothesize that as D-serine levels in the brain peak during wakefulness, synaptic plasticity is heightened. We will use STING to inactivate the release of D-serine from astrocytes to determine the effects on synapses. STING is achieved by fusing miniSOG, a light-sensitive singlet oxygen generator to a critical mediator of vesicle release. Loss of function is achieved by triggering miniSOG with 480nm light to release singlet oxygen which oxidizes amino acid residues and inactivates protein function. We will validate that miniSOG-VAMP2/3 is expressed in the brain following adeno-associated virus (AAV) transduction. We will perform electrophysiology in hippocampal slices, and assess how STING affects the release of the gliotransmitter D-serine. Given that D- serine levels oscillate throughout the day in a vigilant-state (wake versus sleep)-dependent manner, we will examine the contribution of D-serine to synaptic plasticity using STING during wakefulness and during sleep. Aim 1: Spatio-Temporal INactivation of Gliotransmission (STING): Determining the cellular and subcellular localization of the miniSOG-VAMP2/3 construct. Aim 2: Demonstrate that inactivation of miniSOG-VAMP2/3 in either neurons or astrocytes affects synaptic transmission. Aim 3: Determine the astrocytic contribution to synaptic plasticity Using STING, we will gain valuable insight into the astrocytic contribution of D-serine to synaptic plasticity, which has major translational relevance to schizophrenia, where there is a hypofunction of NMDARs. Altering astrocytic gliotransmission may be a viable pathway for modulating NMDARs, which are a major therapeutic target for this neuropsychiatric disease. Achieving light-controlled loss of function will provide unprecedented control in the analysis of protein function in neural circuits and behavior, and can be broadly applied across many fields in biomedical sciences. Completion of the proposed studies will prepare the applicant for a successful career as an independent researcher in the field glial biology.